SUMMARY for TR&D 4 The overarching goal of TR&D 4 is to introduce concepts of precision medicine to chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI). CEST MRI amplifies signal from macromolecular species that contain protons in exchange with water. Currently CEST leverages the suitable chemical shift frequencies and exchange rates of amide or hydroxyl protons. One may use CEST for detection of proteins that naturally contain many such functionalities or for exogenously administered, artificial agents, such as the lysine-rich reporter from TR&D 2. As such CEST is not an inherently targeted (to phenotypically expressed proteins) or precision method. TR&D 4 of the BTRC will generate and disseminate precision CEST agents. We will develop non-metallic, targeted MR agents for receptor imaging with a focus on polymeric species to ensure adequate signal. Currently, about one third of all MRI scans rely on administration of non-specific relaxation-based gadolinium contrast agents to aid in the clinical differentiation of healthy and diseased tissues, however in view of recent concerns about accumulation of gadolinium in brain and bone, there is an increasing demand for non- metallic agents. CEST MRI allows the imaging of low-concentration compounds with the molar sensitivity of MRI and also has the advantages that CEST agents can be designed to be biodegradable. We will synthesize translatable CEST MRI agents for receptor-based imaging together with acquisition and analysis approaches that are optimized for the properties of each individual agent. To accomplish this, in Aim 1, we will conjugate a PSMA targeting ligand to highly sensitive CEST polymers, including dextran polymers as biocompatible agents and salicylic acid polymers as higher sensitivity polymers. We will then optimize pulse sequences for detecting the polymeric agents in terms of exchange rate and chemical shift (Aim 2). The pulse sequences developed in Aim 2 will also be applied to TR&D 2. In Aim 3 we will further proceed to investigate two other clinically relevant receptors, carbonic anhydrase IX (CA-IX), the expression of which has implications for the tumor microenvironment, and Axl tyrosine kinase. CA-IX is an antigen expressed on clear cell renal cell carcinoma (ccRCC), and could provide valuable information for radical nephrectomy and renal cancer surveillance. In Aim 4, we will generate cGMP-grade material of the most promising item in the Center for Translational Molecular Imaging on the pathway to human use. For this step, the agents generated will be tested in preliminary toxicity studies will be performed prior to GLP toxicity testing.